The present invention relates to methods of carrying out reactions and separations, and, especially, to reactions and separations involving tagging.
Interest in expediting the synthesis of organic compounds for use as potential drugs, agricultural agents, catalysts, ligands and other uses has led to the development of a number of methods for synthesis that use xe2x80x9cmixturesxe2x80x9d of organic compounds rather than pure organic compounds. Simple mathematics demonstrates the potential power of mixture synthesis. For example, to execute a parallel (or sequential) n-step synthesis starting from m different starting materials requires nxc2x7m individual reactions with all the attendant equipment (for example, reaction vessels) and manipulations (transfers, workups, chromatography, etc). However, if the m compounds are mixed at the beginning, and then carried through the n-step synthesis and separated, only n separate steps are required.
The value of mixture synthesis has recently been demonstrated in the area of solid phase synthesis with techniques of split synthesis. For example, by using xe2x80x9cone bead/one compoundxe2x80x9d techniques, large libraries of compounds can be made in relatively few steps. See, for example, Lam, K. S., et al., xe2x80x9cThe xe2x80x98One-Bead-One-Compoundxe2x80x99 Combinatorial Library Method,xe2x80x9d Chem. Rev., 96, 411-488 (1996); Thompson, L. A. and Ellman, J. A., xe2x80x9cSynthesis and Applications of Small Molecule Libraries,xe2x80x9d Chem. Rev., 96, 555-600 (1996). Each bead is effectively a kind of reaction vessel that permanently holds its xe2x80x9ccontentsxe2x80x9d (substrates and their products) by chemical bonds. The beads are mixed, not the compounds. Likewise, methods such as using xe2x80x9ctea bagsxe2x80x9d, xe2x80x9cmicrokansxe2x80x9d, and other physical equipment have been introduced to facilitate mixture synthesis. However, in all those solid phase synthesis techniques it is the container of the supported substrates that is mixed. The substrates themselves are polymer-bound and are not mixed. Such solid phase synthesis techniques are typically limited by difficulty in developing suitable reaction conditions for generally biphasic reactions.
Organized mixtures of organic molecules (libraries) have also been generated by using solution phase chemistry. See, for example, Houghten, R. A., xe2x80x9cMixture-Based Synthetic Combinatorial Libraries,xe2x80x9d J. Med. Chem., 42, 3743-3778 (1999). Although such libraries can be made in different ways, a common thread in that approach is that no effort is made to separate the mixture into individual pure components. Instead, libraries and sub-libraries are constructed and assays are conducted such that an active component (or components) can be identified by a process of deconvolution. Deconvolution processes are generally methods which attempt to identify the most active members of a library of compounds without isolating the individual components of the library. In general, mixtures of compounds are tested to measure an average activity of the mixture. Mixtures can be separated by HPLC fractionation or other standard techniques for separation of organic molecules, but the separation typically does not provide pure components since mixture components overlap. See, for example, Griffey, H. Y., xe2x80x9cRapid Deconvolution of Combinatorial Libraries Using HPLC Fractionation,xe2x80x9d Tetrahedron, 54, 4067-4076 (1998). Further, the outcome of the separation (that is, which fractions are pure and which are mixtures, as well as which fraction contains which compound(s)) is not generally known in advance.
It is very desirable to develop improved reaction and separation systems to, for example, enhance the utility of mixture synthesis.
In general, the present invention provides a method of separating compounds that includes the steps of: tagging a first organic compound with a first tagging moiety to result in a first tagged compound; tagging at least a second organic compound with a second tagging moiety different from the first tagging moiety to result in a second tagged compound; and separating the first tagged compound from a mixture including the second tagged compound using a separation technique based upon differences between the first tagging moiety and the second tagging moiety. Preferably, the separation technique is based upon difference in the fluorous nature of the first tagged compound and the second tagged compound, differences in total charge between the first tagged compound and the second tagged compound, differences in size between the first tagged compound and the second tagged compound, and/or differences in polarity between the first tagged compound and the second tagged compound.
As used herein, the term xe2x80x9ctaggingxe2x80x9d refers generally to attaching a moiety or group (referred to as a xe2x80x9ctagging moietyxe2x80x9d or xe2x80x9ctagging groupxe2x80x9d) to a compound to create a xe2x80x9ctagged compoundxe2x80x9d. Preferably, the tagging moiety is attached via covalent bond. However, other strong attachments such as ionic bonding or chelation can also be used. In the present invention, different tagging moieties are preferably used on different compounds to facilitate separation of such tagged compounds.
For example, the tagging moieties can be fluorous moieties that differ in fluorine nature (for example, fluorine content and/or structure). As used herein, the term xe2x80x9cfluorousxe2x80x9d, when used in connection with an organic (carbon-containing) molecule, moiety or group, refers generally to an organic molecule, moiety or group having a domain or a portion thereof rich in carbon-fluorine bonds (for example, fluorocarbons, fluorohydrocarbons, fluorinated ethers and fluorinated amines). The term xe2x80x9cfluorous substrate,xe2x80x9d thus refers generally to a substrate comprising a portion rich in carbon-fluorine bonds. As used herein, the term xe2x80x9cperfluorocarbonsxe2x80x9d refers generally to organic compounds in which all hydrogen atoms bonded to carbon atoms have been replaced by fluorine atoms. The terms xe2x80x9cfluorohydrocarbonsxe2x80x9d and xe2x80x9chydrofluorocarbonsxe2x80x9d include organic compounds in which at least one hydrogen atom bonded to a carbon atom has been replaced by a fluorine atom. The attachment of fluorous moieties to organic compounds is discussed in U.S. Pat. Nos. 5,859,247 and 5,777,121, the disclosures of which are incorporated herein by reference.
Separation of the tagged compounds of the present invention is achieved by using separation techniques that are complementary to (based upon differences between) the tagging moieties. For example, in the case that compounds are tagged with fluorous moieties that differ in fluorine content, the tagged compounds may be separated using a fluorous separation technique (for example, fluorous reverse phase chromatography).
As used herein, the term xe2x80x9cfluorous separation techniquexe2x80x9d refers generally to a method that is used to separate mixtures containing fluorous molecules or organic molecules bearing fluorous domains or tags from each other based predominantly on the fluorous nature of molecules (for example, size and/or structure of the fluorous molecule or domain). Fluorous separation techniques include but are not limited chromatography over solid fluorous phases such as fluorocarbon bonded phases or fluorinated polymers. See, for example, Danielson, N. D. et al., xe2x80x9cFluoropolymers and Fluorocarbon Bonded Phases as Column Packings for Liquid Chromatography,xe2x80x9d J. Chromat., 544, 187-199 (1991). Examples of suitable fluorocarbon bonded phases include commercial Fluofix(copyright) and Fluophase(trademark) columns available from Keystone Scientific, Inc. (Bellefonte, Pa.), and FluoroSep(trademark)-RP-Octyl from ES Industries (Berlin, N.J.). Other fluorous separation techniques include liquid-liquid based separation methods such as countercurrent distribution with a fluorous solvent and an organic solvent.
As indicated above, a number of tagging strategies other than fluorous tagging are suitable for use in the present invention. In general, any tagging strategy that facilitates separation of the tagged compounds based on differences in the tag is suitable. If compounds that are tagged are to undergo one or more reactions to produce tagged product compounds that are to be separated, the tagging moieties preferably do not substantially interfere with the reaction(s) and are not cleaved during the reaction(s). In that regard, the product compounds must be tagged to achieve separation based upon differences in the tagging moiety. As will be discussed further below, the manner/order of steps in which the tagged product compounds become tagged is unimportant.
In addition to tagging moieties that differ in fluorine content, tagging moieties that, for example, differ in total charge can also be used in the present invention. Such tagged compound can, for example, be separated by electrophoresis. The tagging moieties can also be oligomers, polymers, or dendrimers that differ in size. In the case that the tagging moieties are oligimers, polymers or dendrimers, the tagged compounds can, for example, be separated by size exclusion chromatography. As used herein, the terms, xe2x80x9coligomersxe2x80x9d and xe2x80x9cpolymersxe2x80x9d refer generally to molecules that are made by linking together repeating units of one or more small molecules called monomers. Generally, oligomers include fewer monomer units than polymers, although the precise border between an oligomer and a polymer in not well defined. In the present invention, so-called xe2x80x9csolublexe2x80x9d oligomers and polymers are preferred. Soluble polymers are discussed in, for example, Gravert, D. J. and Janda, K. D., xe2x80x9cOrganic Synthes is on Soluble Polymer Supports: Liquid-phase Methodologies,xe2x80x9d Chem. Rev., 97, 489-509 (1997). Through use of soluble oligimer or polymer tags, substrates or products can be attached to oligomer or polymer tags of different molecular weights or molecular weight ranges and then the tagged substrates can be mixed to generate a true mixture which can, if desired, be reacted in standard solution phase organic reactions prior to separation. An example of a suitable family of oligomer/polymer tags is polyethylene glycol (PEG, H(OCH2CH2)nOH). PEG is soluble in an assortment of organic solvents, has two terminal hydroxyl groups for attachment/detachment of compounds and products, and can be purchased in a range of sizes (for example, average molecular weights of 1,000, 1,500, 2,000, 4,600, etc).
As used herein, the term xe2x80x9cdendrimerxe2x80x9d refers generally to branched or hyperbranched molecules that are synthesized in generations by attachments of successive sets of building blocks to a core (or the inverse). See, for example, Dendrimers, F. Vogtle, Ed., Springer-Verlag Berlin: Heidelberger Platz 3/W-1000 Berlin 33/Germany, 1-18 (1998). Unlike traditional oligomers and polymers, dendrimers can be made largely as pure molecules each bearing the same number of building blocks. To the contrary, oligomers and polymers are generally available only as mixtures of molecules with a distribution of sizes centered around an average. Different generations of dendrimers make convenient families of tags. For example, the various generations of the commercially available xe2x80x9cDABxe2x80x9d polypropylene amine dendrimers (tetraamine, octaamine, hexadecamine, etc.) vary widely in size and molecular weight and provide increasing numbers of amines for attachment. Starburst(copyright) (PAMAM) dendrimers provide another example of a family of dendrimers tags.
The tagging moieties can also differ in polarity, in which case the tagged compounds can, for example, be separated by standard or, preferably, reverse phase chromatography.
The present invention also provides a method for carrying out a chemical reaction including the steps of: tagging a plurality of compounds with different tagging moieties to create tagged compounds, conducting at least one chemical reaction on the tagged compounds to produce a mixture of tagged products, and separating the mixture of tagged products by a separation technique based upon differences in the tagging moieties. The method may further include the step of removing the tagging moieties from the tagged products after separation. The tagging moieties are removable using standard reactions as known in the art. The tagging strategies described above can be used.
The order or sequence of steps in which the tagged products are produced is unimportant. In one embodiment of the present invention, for example, compounds are tagged and then mixed. In other embodiments, the tagging step itself generates the tagged compound mixture. In still other embodiments, the compounds are already mixed prior to the tagging. In these embodiments, each tag is preferably attached to a single compound by using selective reactions known to those skilled in the art. In general, selective reactions are those in which one component or subset of components of a mixture reacts faster than another component or subset of components. Selective reactions can, for example, be based on differences in reacting functional groups, steric effects, electronic effects, and stereoelectronic effects, among other things. For example, if a mixture contains a secondary and a primary alcohol, it is possible to use the higher reactivity of the primary alcohol to selectively attached a first tag to it, and then attach a second tag to the remaining secondary alcohol. If a mixture contains two enantiomers, it is possible to selectively tag one enantiomer with a first tag using a chiral catalyst or reagent, and then tag the remaining enantiomer with a second tag. In these types of reactions, it is preferable that each tagging reaction be selective for its target component(s) of the mixture to a level of at least approximately 80%. More preferably, the level of selectivity is at least approximately 90%.
The present invention is particularly useful in conjunction with mixture synthesis or combinatorial synthesis. It is thus beneficial to briefly discuss a number of terms commonly used in such synthetic schemes. As used herein, the term xe2x80x9csubstratexe2x80x9d refers generally to a reaction component that is a starting material of a synthetic reaction, normally purchased prepared in a prior step. The terms xe2x80x9cproductxe2x80x9d or xe2x80x9ctarget productxe2x80x9d refer generally to the target or desired molecule(s) of a transformation derived by reaction of the substrate with the other reaction component(s) in a reaction medium. The terms xe2x80x9cside productxe2x80x9d or xe2x80x9cbyproductxe2x80x9d refer generally to a product derived from any component(s) of the reaction medium which is not the target product and is preferably separated therefrom.
The term xe2x80x9creagent,xe2x80x9d as used herein refers generally to a chemical entity that is required for a reaction but contributes either an invariant piece or no piece to the products of a mixture synthesis or a combinatorial synthesis. The term xe2x80x9creactant,xe2x80x9d as used herein refers generally to a type of molecule that contributes a variable piece to the products of a mixture synthesis or a combinatorial synthesis. The distinction between the terms xe2x80x9creactantxe2x80x9d and xe2x80x9creagentxe2x80x9d in xe2x80x9ccommonxe2x80x9d (non-mixture and non-combinatorial) organic syntheses is vague, but those skilled in the art often refer to a reaction component as a reagent if it contributes no piece, a rather small piece, or a piece without carbon atoms therein to the target product. As used herein, the term xe2x80x9creagentxe2x80x9d includes a catalyst if used in a substoichiometric quantity. Both substrates and reactants are sometimes referred to as starting materials or starting compounds.
In common organic synthesis, individual steps are conducted sequentially until the final target molecule or product is made. In combinatorial organic synthesis, the target is not a single molecule but instead a xe2x80x9clibraryxe2x80x9d of several to millions of molecules. Combinatorial synthesis can be carried out by parallel synthesis of individual pure compounds or synthesis of mixtures.
Once again, the manner in which a product compound is tagged in a mixture synthesis or combinatorial synthesis in the present invention is unimportant. For example, a tagging moiety can be incorporated into a substrate, reactant and/or reagent to create a tagged product.
Likewise, individual or mixed, untagged product compounds can be directly tagged with tagging moieties to create tagged product compounds. It is important that the different products or subsets of products have different tags.
In mixture and combinatorial synthesis, multiple reactions are conducted either together or in parallel to provide multiple products. In mixture synthesis and combinatorial synthesis, the premium of simple methods of purification is even higher than in normal synthesis. For this reason, combinatorial synthesis is now commonly conducted on the solid phase, where purification can be effected simply by filtration. However, conducting such reactions can be difficult because the solid-bound reaction component never truly dissolves in the reaction solvent.
There are a number of advantages afforded by carrying out mixture or combinatorial synthesis in a liquid phase as enabled by the present invention. For example, many reactions are preferably conducted in a homogenous liquid phase. This is in direct contrast to solid phase syntheses, where true homogeneity is never obtained.
Moreover, unlike deconvolution methods, the methods of the present invention can be used to readily provide pure product compounds for testing. In the case of deconvolution methods, mixtures of product compounds are tested to identify the highest average activity. Much valuable information can be lost in such deconvolution methods. For example, even testing of non-optimal (with respect to biological activity, for example) pure compounds provides valuable information.
Furthermore, unlike standard separation techniques, the separation of a mixture of tagged compounds in the present invention is determined primarily by the nature of the tag rather than the component/compound that is tagged. Preferably, the tagging compounds of the present invention are chosen such that they are separable in a predetermined manner via a complementary separation technique. For example, compounds of different fluorine content will separate in a predetermined order during chromatography over solid fluorous phases. Because compounds are preferably selectively tagged with certain tagging moieties in the present invention, the manner (for example, order) in which specific tagged compounds will separate is predetermined, thereby potentially eliminating the need to chemically identify the results of the separation.
In general, the present invention provides substantially universal methods for synthesizing and separating organic compounds. The methods are particularly useful in mixture and combinatorial synthesis techniques, but find use in substantially any reaction and/or separation requiring separation of one organic compound from another organic compound.
Typically, known standard (non-tagged) reactions can be carried out under the present invention with one or more tagged compounds within the range of reaction conditions used in the corresponding standard (non-tagged) reactions. The present invention is equally applicable, however, to newly developed organic reactions.
Transformations under the method of the present invention thus generally parallel the transformations of known xe2x80x9cnon-taggedxe2x80x9d substrates with the advantages that the tagged products are more easily separated from untagged reaction components and from each other.